Liberty Sibanda

Lateral flow colloidal gold based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges

A lateral-flow immunoassay using a colloidal gold-labelled monoclonal antibody was developed for the rapid detection of deoxynivalenol (DON). Different parameters, such as the amount of immunoreagents, type of the materials, composition of the blocking solution and of the detector reagent mixture, were investigated to provide the optimum assay performance. The experimental results demonstrated that such a visual test had an indicator range rather than a cut-off value. Thus, tests for DON determination with two different indicator ranges of 250-500 and 1000-2000 microg kg(-1) were designed. The method allowed detection of DON at low and high concentration levels, which could be useful for research and practical purposes. The assay applied to spiked wheat and pig feed samples demonstrated accurate and reproducible results. The applicability of the developed lateral-flow test was also confirmed under real field conditions. The test strips prepared in Belgium were sent to Mexico, where they were used for the screening of DON contamination in different bread wheat entries from Fusarium Head Blight inoculated plots. The results were compared with those obtained by ELISA and LC-MS/MS. A poor correlation between ELISA and LC-MS/MS was observed. Visual results of the dipstick tests were in a good agreement with the results of the LC-MS/MS method. Coupled with a simple and fast sample preparation, this qualitative one-step test based on the visual evaluation of results did not require any equipment. Results could be obtained within 10 min. The described assay format can be used as a simple, rapid, cost-effective and robust on-site screening tool for mycotoxin contamination in different agricultural commodities.

A collaborative study to validate novel field immunoassay kits for rapid mycotoxin detection.

Kits designed to detect ochratoxin A (OA) and T-2 toxin by a membrane-based flow-through enzyme immunoassay were studied collaboratively by screening cereals (wheat, rye, maize and barley) for the presence of these mycotoxins. Sample preparation and test procedure were clearly described in the instruction leaflets included in the kits. A simple methanol-based extraction followed by filtration and dilution steps was prescribed. Reagents were successively pipetted to the membrane of the device, then colour development was evaluated visually. Limits of detection for the ochratoxin A and T-2 toxin tests were 4 and 50 microg kg(-1), respectively. Five laboratories took part in the first stage of this study, and five more joined the second stage. Cereal samples (blank, spiked or inoculated) were shipped with the kits to the participating laboratories, while results obtained were confirmed by high-performance liquid chromatography with fluorescence detection and by gas chromatography-mass spectrometry for ochratoxin A and T-2 toxin, respectively. Some initial difficulties were encountered. In the second stage, four ochratoxin A and four T-2 toxin kits were used by 10 collaborators to analyse 21 cereal samples. For the ochratoxin A kits, the percentage of false positive and false negative results were 2% and 4%, respectively. The results of one T-2 toxin kit were outliers and when excluded, the overall percentage false positive and false negative results were 6% and 3%, respectively.

Development of a portable field immunoassay for the detection of aflatoxin M1 in milk.

Many methods for AFM1 detection exist, but most are time consuming, employ expensive equipment and require experienced personnel. To overcome these problems a membrane-based flow-through enzyme immunoassay has been developed (patent pending). The assay comprised a nylon Immunodyne ABC membrane spotted with anti-mouse antibodies, a plastic snap-fit device, absorbent cotton wool, mouse anti-AFM1 monoclonal antibodies (Mab), and AFB1-horseradish peroxidase (HRP) conjugate. This assay was coupled to an immunoaffinity column (IAC). The visual detection limit was 0.05 ng/g AFM1 in milk. Assay time for IAC clean-up was 12 min, and that for the flow-through assay was 18 min, hence the total assay time was 30 min. This method allows for a rapid screening of milk consignments which do not conform to the maximum permissible limits of 0.05 ng/g AFM1, hence enabling the rejection of such at the farm level. Laboratory validation was done using certified reference materials (CRM) with AFM1 concentrations of < 0.05, 0.09 and 0.76 ng/g. Precision of the assay was high as shown by the high repeatability of the assay results. There were no significant differences in recoveries between standard in buffer and CRM (P > 0.05), and assay responses for these two were highly correlated (99.63%).

Optimization of solid-phase clean-up prior to liquid chromatographic analysis of ochratoxin A in roasted coffee.

An extraction and clean-up method for ochratoxin A (OA) in roasted coffee has been developed and the HPLC method optimized. An interfering compound with a similar retention time as OA was adsorbed by the aminopropyl (NH2) material at < or = 5% NaHCO3. Residual OA on the column was recovered by washing with the extraction solution followed with methanol. Fractions were mixed together for further clean-up with Ochratest immunoaffinity columns (IACs). Analysis by HPLC resulted in a well resolved OA peak and reduction in matrix interferences. Recoveries ranged from 72 to 84% and the detection limit was 1 ng/g.

Review of mycotoxin work in sub-Saharan Africa

Abstract In Sub-Saharan Africa, work on mycotoxins covering field cases, acute exposures and chronic effects related to dietary intake is reviewed. Mycotoxins have been implicated in the etiology of diseases like kwashiorkor, marasmic kwashiorkor, hepatocellular carcinoma in humans, and hepatocardnomas, encephalopathy and other acute diseases in animals. Mycotoxin regulatory programmes using maximum permissible limits of the different mycotoxins are also summarized. Maximum permissible levels of mycotoxins in most countries have been considered for aflatoxins, and progress in mycotoxin research indicates that, soon, more mycotoxins will be included.

Survey of Fusarium moniliforme (F. verticillioides) and production of fumonisin B1 in cereal grains and oilseeds in Zimbabwe

In a national survey carried out in 1995 and 1996, the distribution of Fusarium moniliforme (= F. verticillioides) and fumonisin B1 (FB1) levels in cereals and oilseeds from Zimbabwe were analyzed. The results of this study showed that the incidence of F. moniliforme and other Fusarium species and levels of FB1 generally decreased from regions with high rainfall and annual moderate temperatures to low rainfall regions. There was no Fusarium contamination and FB1 was not detected in sunflowers and soybeans. The incidence of F. moniliforme and its metabolite exhibited a substrate preference with high incidences of Fusarium species and high FB1 levels being recorded for maize followed by wheat, rapoko and sorghum.

Development of a flow-through enzyme immunoassay and application in screening green coffee samples for ochratoxin A with confirmation by high-performance liquid chromatography.

A flow-through enzyme immunoassay has been developed for the screening of green coffee bean samples for ochratoxin A (OA) and was later used in a survey on OA in green coffee from different countries. The test has a sensitivity of 8 ng/g, and calculated recoveries ranged from 70 to 89% and from 86 to 95% for spiked and naturally contaminated samples, respectively. There were no significant differences in within-day and between-day assay performance (P > 0.05). Green coffee samples (15 Arabica and 7 Robusta) received from an international coffee trader were analyzed for intrinsic fungal contamination, screened for OA, and subsequently confirmed by high-performance liquid chromatography (HPLC). All 22 samples were contaminated by fungal species of the genus Aspergillus, while Penicillium species were isolated from a mere 13.6% of the total number of samples. Isolates were tested for their ability to produce OA, and only 3.9% were positive. There was no correlation between occurrence of OA-producing isolates and levels of OA in contaminated samples. Results of the screening procedure showed that 4 of the 22 samples were contaminated with 8 ng/g or higher. The HPLC method confirmed that the OA levels ranged from 27 to 168 ng/g. A fifth sample, which was shown to be negative during screening, had an OA concentration of 4 ng/g. There were no false negatives or positives recorded, and the flow-through enzyme immunoassay results correlated with those obtained by HPLC.

Application and validation of a clean-up tandem assay column for screening ochratoxin A in cocoa powder

A rapid antibody-based assay for the detection of ochratoxin A in cocoa powder is described, involving sequential clean-up and visual detection of the toxin (“clean-up tandem assay column”). The screening test was developed to have a cut-off level of 2 µg kg−1 and was shown to have false positive and false negative rates of 10 and 2%, respectively. Analysis of six samples can be carried out in the field in approximately 30 min by untrained workers. Using the proposed rapid screening test, 10 retail cocoa powders were found to contain no detectable levels of ochratoxin A (<2 µg kg−1). These samples were also found to be negative (<2 µg kg−1) when analysed using an LC–MS/MS method.

Novel developments in rapid mycotoxin detection

Rapid antibody-based mycotoxin screening techniques are designed to be used outside a laboratory environment, at the place of sampling. Results are expected immediately, so that commodities can be further processed without delay. Because they are used for mycotoxin analysis, very low levels (ppb and ppt range) should be detected. A further requirement is that the obtained results are accurate with a false negative rate of <5% at the level of interest.At first, plastic microtiter plates were used as solid phase materials for immobilizing antibodies (enzyme-linked immunosorbent assays). However, to increase speed and user-friendliness, plastics were replaced by microporous membranes. As an example a flow-through enzyme immunoassay for the detection of fumonisins in cornflakes with a cut-off value of 275 μg/kg is described. No false negative results were observed and the false positive rate was 18%. However, enzyme labels, used to enable visual evaluation of results, did not seem to be completely satisfactory in terms of stability and repeatability of the generated signal. Therefore microparticle labels such as colloidal gold particles are used more and more,e.g. in a lateral flow dipstick immunoassay. When applied to the detection of aflatoxin B1 in pig feed a cut-off value of 5 μg/kg could be reached with no false negative results and a false positive rate of only 10%. Sample pretreatment for screening techniques should be rapid and simple. Preferably a simple solvent extraction is used, followed by a filtration and dilution step. However, for strongly coloured or complex food matrices, this did not seem to work. The combination of clean-up and detection in one single test device is a new approach. When using this clean-up tandem assay column for the detection of ochratoxin A in roasted coffee, a cut-off value of 6 μg/kg was reached. No false positive results were obtained, however, the false negative rate was 8%.